Novel protein, a gene encoding therefor and a method of using the same

ABSTRACT

An object of the present invention is to search and identify novel antifungal proteins capable of inhibiting the growth of plant pathogenic microorganisms including  Magnaporthe grisea  and  Rhizoctonia solani  causing two major rice diseases at relatively low concentrations, and further to clone a gene for said protein. The present invention provides an antifungal protein which can be obtained from fraction(s) precipitated by ammonium sulfate precipitation using an aqueous extract from  Pleurotus cornucopiae , wherein said protein has an antifungal activity against at least rice blast, and exhibits existence of a component having a molecular weight of about 15 kDa as determined by SDS-PAGE method; a gene encoding said protein and uses thereof.

This application is a Divisional of co-pending application Ser. No.12/716,182, filed on Mar. 2, 2010 and for which priority is claimedunder 35 U.S.C. §120, which is a Divisional of application Ser. No.12/369,587, filed on Feb. 11, 2009 and for which priority is claimedunder 35 U.S.C. §120, which is a Divisional of application Ser. No.10/471,422 filed on Sep. 11, 2003, and for which priority is claimedunder 35 U.S.C. §120, which is the National Stage of PCT/JP02/02295filed on Mar. 12, 2002, which claims the benefit of priority of JapaneseApplication No. 2001/68894 filed on Mar. 12, 2001 under 35 U.S.C. §119.The entire contents of application Ser. No. 12/716,182, filed on Mar. 2,2010, application Ser. No. 12/369,587, filed on Feb. 11, 2009,application Ser. No. 10/471,422 filed on Sep. 11, 2003, PCT/JP02/02295,filed on Mar. 12, 2002, and Japanese Patent Application No. 2001/68894,filed on Mar. 12, 2001 are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel protein having an antifungalactivity and a method for producing thereof, a gene encoding theprotein, and a method of using the protein and gene. Specifically, itrelates to a protein originated from Pleurotus cornucopiae having anantifungal activity against at least rice blast (Magnaporthe grisea), agene encoding the protein, and a method of using the protein and gene.

The present application claims priority based on Japanese PatentApplication No. 2001-68894 filed on Mar. 12, 2001, the entire contentsof which are incorporated herein as a reference.

BACKGROUND ART

Lytic enzymes such as chitinase and β-1,3-glucanase are known as plantproteins having an antifungal activity against plant pathogenicmicroorganisms. In vitro experiments have shown that these enzymes canexert the effect if employed alone (Schlumbaum et al. (1986), Nature324, pp. 365-367), but enhanced effect can generally be obtained if acombination of two or more of such enzymes is used (Mauch et al. (1988),Plant Physiol. 88, pp 936-942). It is known that the growth inhibitionconcentration of these lytic enzymes against filamentous fungi should betypically about several tens to several hundreds of μg/ml when usedalone, or about several μg/ml per enzyme when used in combination.However, none of these lytic enzymes has been reported to have anantifungal effect against rice blast (Magnaporthe grisea), which causesextensive damage to rice crops.

Antifungal peptides (AFP) of low-molecular weight such as defensin alsohave an antimicrobial activity. Among them, Ca-AMP1 (Japanese DomesticAnnouncement No. 505048/96), CB-1 (Oita et al. (1996), Biosci. Biotech.Biochem. 60, pp. 481-483), Rs-AFP1 and Rs-AFP2 (Terras et al. 1992, J.Biol. Chem. 267, pp. 15301-15309), and Ace-AMP1 (Japanese DomesticAnnouncement No. 501424/97) have been reported to have an antifungaleffect against rice blast. These low-molecular weight peptides inhibit50% of the growth of various plant pathogenic microorganisms includingthe one mentioned above at a concentration in the order of severalμg/ml.

Attempts have also been made to create a disease-resistant plant byisolating the gene for a lytic enzyme or a low-molecular weightantifungal peptide and transfecting it into a plant (Broglie et al.(1991), Science 254, pp. 1194-1197; Terras et al. (1995), The Plant Cell7, pp. 573-588). A recent study of rice reported that transformant riceobtained by overexpressing rice-derived chitinase exerted increased riceblast resistance (Nishizawa et al. (1999) Theor. Appl. Genet.99:383-390).

Other pathogenic microorganism-resistant plants created by geneintroduction have also been reported such as for PR protein (Alexanderet al. (1993) Proc. Natl. Acad. Sci. USA 90: pp. 7327-7331), glucoseoxidase (Wu et al. (1995) Plant Cell 7: pp. 1357-1368), stilbenesynthase (Hain et al. (1993) Nature 361: pp. 153-156), etc.

However, many existing cases fail to obtain transgenic plants havingpractically acceptable resistance. This may be attributed to the lowexpression level of the transgenes, and more essentially the lowantifungal activity of the antifungal proteins so far reported.Therefore, it would be desirable to identify and practically apply amore potent antifungal protein than conventional ones.

DISCLOSURE OF THE INVENTION

An object of the present invention is to search and identify a novelantifungal protein capable of inhibiting the growth of various plantpathogenic microorganisms, including rice blast (Magnaporthe grisea),which causes extensive damage to rice crops.

Another object of the present invention is to clone a gene encoding saidnovel protein, and to determine the nucleotide sequence thereof.

Still another object of the present invention is to introduce the geneof the present invention into a host organism (microorganism, animal,plant, etc.) to create a transformant, and thereby put to practical usethe gene of the present invention.

Still another object of the present invention is to provide anantifungal agent containing the antifungal protein of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows appearance of the growth inhibition of M. grisea by theantifungal protein of the present invention (48-hour cultures in thepresence of a protein fraction heated at 80° C. for 10 minutes).

FIG. 2 shows electrophoretic patterns of Pleurotus cornucopiae proteinfractions separated by a Q-Sepharose FF column in relation to anantifungal activity. M represents a molecular weight marker, and FTrepresents the fraction having passed through the column. The symbols(−, +, ++) below the lanes indicate the strength of the antifungalactivity. The antifungal activities shown in parentheses belong tofractions other than the purified ones described in the presentinvention.

FIG. 3 shows a separation chart of the Pleurotus cornucopiae antifungalprotein by MonoQ column in relation to an antifungal activity. Theelution positions showing the antifungal activity are indicated by +.

FIG. 4 shows electrophoretic patterns of Pleurotus cornucopiae proteinseparated by a Mono Q column in relation to an antifungal activity. Thenumbers above the lanes correspond to the fraction numbers in FIG. 3, Mrepresents a molecular weight marker, and On represents the Q-Sepharosefraction applied on Mono Q. The symbols (−, +, ++, +++) below the lanesindicate the strength of an antifungal activity. The arrows indicate theantifungal protein (15 kDa).

FIG. 5 shows a separation chart of the Pleurotus cornucopiae antifungalprotein by Superose 6 in relation to an antifungal activity. The arrowsindicate the elution positions of Gel filtration standard (BIO-RAD). Thepositions of fractions showing the antifungal activity are indicated by+.

FIG. 6 shows electrophoretic patterns of Pleurotus cornucopiae proteinpurified by Superose 6 in relation to an antifungal activity. Thenumbers above the lanes correspond to the fraction numbers in FIG. 5,Ori represents the MonoQ fraction applied on Superose 6, and Mrepresents a molecular weight marker. The symbols (−, +, ++) below thelanes indicate the strength of an antifungal activity. The arrowindicates the antifungal protein (15 kDa).

FIG. 7 shows a molecular taxonomic tree of the amino acid sequences(mature protein regions) of tamavidin 1 and tamavidin 2, streptavidinand its homolog, and avidin.

FIG. 8 shows the results of experiments of abolishment of an antifungalactivity by addition of biotin. Spores of M. grisea suspended in ½ PDwere placed in microtiter plates. Wells containing 1000 ng/ml ofpurified tamavidin 1, streptavidin and avidin, or wells containing 100ng/ml biotin in addition to these proteins at the same concentrations,or wells containing no protein were prepared and incubated at 28° C. for48 hours. Wells containing 50 ng/ml of purified tamavidin 1 are alsoshown.

FIG. 9 shows purification of recombinant tamavidin 2 expressed in E.coli on an iminobiotin column. C represents the total soluble proteinfraction of E. coli, which was not induced by IPTG, and T represents thetotal soluble protein fraction of E. coli induced by 1 mM IPTG. Frepresents the protein fraction having passed through the iminobiotincolumn without binding to the column, obtained from the total solubleprotein fraction of E. coli induced by 1 mM IPTG, W represents thefraction eluted by washing the column, and E represents the fractioneluted with an acidic buffer. Arrows indicate tamavidin 2 protein (about15 kDa), and M represents a molecular weight marker (LMW marker kit:Pharmacia LKB).

DETAILED DESCRIPTION OF THE INVENTION

With the purpose of solving the problems described above, the presentinventors first established an assay system for evaluating an in vitroantifungal activity against rice blast.

Then, protein fractions were extracted from an edible mushroom Pleurotuscornucopiae and subjected to the antifungal assay to identify antifungalprotein fractions and isolate and purify an antifungal protein bycombining ion exchange chromatography and gel filtration. Partial aminoacid sequences of the purified protein were determined, on the basis ofwhich oligo DNA sequences were synthesized, and then a partial lengthcDNA encoding the protein was obtained by RT-PCR. Then, a cDNA libraryof Pleurotus cornucopiae fruit body was screened by using the partiallength cDNA as a probe to identify a full-length cDNA encoding theprotein, and the total nucleotide sequence thereof was determined. Thus,the total amino acid sequence of the Pleurotus cornucopiae antifungalprotein and the nucleotide sequence of a gene encoding thereof wereidentified, thereby completing the present invention.

Accordingly, a first aspect of the present invention provides anantifungal protein which can be obtained from fraction(s) precipitatedby ammonium sulfate precipitation method using an aqueous extract fromPleurotus cornucopiae, wherein said protein has an antifungal activityagainst at least rice blast, and exhibits existence of a componenthaving a molecular weight of about 15 kDa as determined by SDS-PAGEmethod.

The antifungal protein of the present invention is typicallycharacterized by the sequence of 143 amino acids shown in SEQ ID NO: 2in the Sequence Listing attached hereto. This protein comprises a unitof a polypeptide having a molecular weight of about 15 kDa as estimatedby SDS-PAGE (corresponding to a polypeptide consisting of amino acids8-143 in the sequence of SEQ ID NO: 2 in the Sequence Listing). Thisprotein was also identified as a protein characterized by a molecularweight of about 30 kDa as determined by gel filtration column.

The antifungal protein of the present invention also includes a proteinhaving 141 amino acids shown in SEQ ID NO: 4 in the Sequence Listing.The protein having the amino acid sequence of SEQ ID NO: 4 alsocomprises a unit of a polypeptide having a molecular weight of about 15kDa as estimated by SDS-PAGE and has a molecular weight of about 30 kDaas determined by gel filtration column, similar to the protein havingthe amino acid sequence of SEQ ID NO: 2.

The antifungal protein of the present invention includes antifungalproteins having not only the amino acid sequence of SEQ ID NO: 2 or 4,but also an amino acid sequence containing one or more amino acidmodifications compared with the original sequence or an amino acidsequence having a homology of 52% or more to the original sequence andshowing an antifungal activity against rice blast.

The antifungal protein of the present invention preferably has an aminoacid sequence having a homology of 52% or more, more preferably 60% ormore, still more preferably 70% or more, further more preferably 80% ormore, especially 90% or more, most preferably 95% or more to the aminoacid sequence of SEQ ID NO: 2 or 4 in the Sequence Listing.

The definition of the “protein having a homology of 52% or more” to eachspecific amino acid sequence as referred to the antifungal protein ofthe present invention means that it may have a homology of at least 52%,preferably 60% or more, more preferably 70% or more, still morepreferably 80% or more, especially 90% or more, most preferably 95% ormore.

A second aspect of the present invention provides an antifungal proteincomprising either one or a combination of a polypeptide consisting of apartial amino acid sequence of SEQ ID NO: 2 or 4 in the SequenceListing, e.g. a polypeptide consisting of a partial amino acid sequence8-143 of SEQ ID NO: 2 or a partial amino acid sequence 8-141 of SEQ IDNO: 4; and a polypeptide having an amino acid sequence containing one ormore amino acid changes in any one of said amino acid sequences or apolypeptide having a homology of 52% or more to any one of said aminoacid sequences and showing an antifungal activity against rice blast.

A third aspect of the present invention provides a method for producingthe antifungal protein of the present invention, which comprises:

collecting fraction(s) from aqueous extract from Pleurotus cornucopiae,precipitated by ammonium sulfate precipitation method using 75%saturated ammonium sulfate; and

applying said fraction(s) to an ion-exchange column chromatography tocollect fraction(s) eluted by NaCl at a concentration between 50 mM-600mM NaCl.

A fourth aspect of the present invention provides a gene encoding theantifungal protein of the present invention.

The gene of the present invention typically has a nucleotide sequenceconsisting of bases 71-502 of SEQ ID NO: 1 or a nucleotide sequence ofbases 226-651 of SEQ ID NO: 3 (hereinafter sometimes simply referred toas “the nucleotide sequence of SEQ ID NO: 1 or 3”), or a nucleotidesequence containing a substitution, deletion, insertion and/or additionof one or more bases in said nucleotide sequence, or a nucleotidesequence hybridizing to said nucleotide sequence in stringentconditions.

The gene of the present invention generally has a nucleotide sequencepreferably having a homology of 60% or more, more preferably 70% ormore, still more preferably 80% or more, especially 90% or more, mostpreferably 95% or more to the nucleotide sequence of bases 71-502 of SEQID NO: 1 or the nucleotide sequence of bases 226-651 of SEQ ID NO: 3.

A fifth aspect of the present invention provides an oligonucleotide forobtaining an antifungal protein from Pleurotus cornucopiae, produced bya method, which comprises:

selecting two regions from a base sequence of a gene encoding theantifungal protein of SEQ ID NO:1 based on the following requirements;

1) length of each regions is 15-30 bases;

2) proportion of G+C content in a base sequence of each region is40-60%;

preparing a single-stranded DNA having a base sequence which isidentical to said region or complementary to said region, or preparingmixture of single-stranded DNAs based on degeneracy of the genetic codewithout changing a sequence of amino acid residues encoded by saidsingle-stranded DNAs; and

optionally preparing a modified version of said single-stranded DNAs,said modification not altering a binding specificity of thesingle-stranded DNAs to the base sequence of the gene encoding saidantifungal protein.

The oligonucleotide of the present invention preferably has thenucleotide sequence of any one of SEQ ID NOs: 10 to 17 in the SequenceListing.

A sixth aspect of the present invention provides a method for isolatingthe gene of the present invention, which comprises performing a nucleicacid amplification reaction using two kinds of oligonucleotidesdescribed above as a pair of primers and cDNA library of Pleurotuscornucopiae fruit body as a template to amplify a portion of the geneencoding the antifungal protein of the present invention, and screeningsaid cDNA library by using thus obtained amplification product as aprobe to isolate the full-length cDNA clone.

A seventh aspect of the present invention provides a recombinant vectorcomprising the gene of the present invention.

As for the recombinant vector of the present invention, the vector ispreferably an expression vector.

An eighth aspect of the present invention provides a transformantobtained by introducing the recombinant vector of the present inventioninto a host organism.

A ninth aspect of the present invention provides an antifungal agentcomprising the antifungal protein of the present invention as an activeingredient.

Preferred Embodiments of the Invention

Preferred embodiments are described in detail below to explain thepresent invention.

Antifungal Protein Derived from Pleurotus cornucopiae

According to a first aspect of the present invention, a protein derivedfrom Pleurotus cornucopiae having an antifungal effect against plantpathogenic microorganism is provided. The protein of the presentinvention is not limited to any specific origin or preparation processso far as it has characteristics defined herein that is, the antifungalprotein of the present invention may be naturally occurring or expressedfrom recombinant DNA by genetic engineering techniques or chemicallysynthesized.

The protein of the present invention typically has the sequence of 143amino acids shown in SEQ ID NO: 2 or the sequence of 141 amino acidsshown in SEQ ID NO: 4 in the Sequence Listing. However, it is well knownthat natural proteins include variant proteins having one or more aminoacid modifications resulting from differences in varieties of theorganisms producing the protein, or the gene mutation depending ondifferences in ecotypes or the presence of closely similar isozymes. Asused herein, the term “amino acid modification” means substitution,deletion, insertion and/or addition of one or more amino acids. Thepresent invention includes the protein having the amino acid sequencesshown in SEQ ID NO: 2 or 4 presumed from the nucleotide sequences of thecloned genes, but it is not restricted thereto. Namely, it is intendedto encompass all homologous proteins having characteristics definedherein. The homology is at least 52% or more, more preferably 60% ormore, still more preferably 70% or more, further more preferably 80% ormore, especially 90% or more, most preferably 95% or more.

Generally, a modified protein containing a substitute from one toanother amino acid having similar properties (such as a substitute froma hydrophobic amino acid to another hydrophobic amino acid, a substitutefrom a hydrophilic amino acid to another hydrophilic amino acid, asubstitute from an acidic amino acid to another acidic amino acid or asubstitute from a basic amino acid to another basic amino acid) oftenhas similar properties to those of the original protein. Methods forpreparing such a recombinant protein having a desired modification usinggenetic engineering techniques are well known to those skilled in theart and such modified proteins are also included in the scope of thepresent invention. For example, site-specific mutagenesis described inMolecular Cloning, 2nd edition (Sambrook et al., (1989)) can be used.

As used herein, the percent homology can be determined by comparisonwith sequence information using the BLAST program described by Altschulet al. (Nucl. Acids. Res. 25, pp. 3389-3402, 1997), for example. Thisprogram is available from the website of National Center forBiotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ) on theInternet. Various conditions (parameters) for homology searches with theBLAST program are described in detail on the site, and searches arenormally performed with default values though some settings may besomewhat changed. Alternatively, it can be determined by comparison withsequence information using a genetic sequence analysis software programsuch as GENETYX (Software Development Co., Ltd.) or DNASIS (HitachiSoftware Engineering).

Homology searches were performed through GenBank databases using BLASTfor the Pleurotus cornucopiae-derived antifungal protein of the presentinvention, and the gene thereof as well as their homologs and proteinshaving an amino acid sequence encoded thereby. Database searches for theamino acid sequence of the first Pleurotus cornucopiae-derivedantifungal protein of the present invention (total amino acid sequenceof SEQ ID NO: 2) revealed matches to streptavidin v2 of Streptomycesviolaceus (Accession No: Q53533, Bayer et al. (1995) Biochim BiophysActa 1263: pp. 60-66), streptavidin v1 (Accession No: □53532),streptavidin of Streptomyces avidinii (Accession No.: P22629, Argaranaet al. (1986) Nucleic Acids Res 14: pp. 1871-1882), etc. The homology ofthese three sequences extends over 128 amino acids, and was 50%, 49% and49%, respectively. A homology of 51.7% to a core streptavidin mutantw79f (Chain B) (Freitag et al. (1997) Protein Sci. 6: pp. 1157-1166) wasalso shown over 120 amino acids.

Egg white avidin (Gope et al. (1987) Nucleic Acids Res 15: pp.3595-3606) and several avidin-related proteins (Keinanen et al. (1994)Eur J Biochem 220: pp. 615-621) were also matched at lower homologydegrees. These facts indicate that the present protein is a novelprotein.

Database searches of the amino acid sequence of the second Pleurotuscornucopiae-derived antifungal protein of the present invention (totalamino acid sequence of SEQ ID NO: 4) showed homology of 50%, 48% and 48%to streptavidin v2, v1 and streptavidin, respectively.

The present antifungal protein was named “tamavidin” because it was anovel streptavidin-like protein purified from an edible mushroomPleurotus cornucopiae (Tamogitake). Here, the gene derived from thepurified protein is called tam1, the protein having an amino acidsequence encoded thereby is called tamavidin 1, a homolog of tam1 iscalled tam 2, and the protein having an amino acid sequence encodedthereby is called tamavidin 2.

The amino acid residues 1-7 of SEQ ID NOs: 2 and 4 are thought tocorrespond to the leader peptide of a precursor of the antifungalprotein. Thus, the amino acid residues 8-143 of SEQ ID NO: 2 and 8-141of SEQ ID NO: 4 are matured forms of the antifungal protein.Accordingly, the present invention also provides an antifungal proteincomprising either one or a combination of a polypeptide consisting of apartial amino acid sequence of SEQ ID NO: 2, that is amino acids 8-143,or a partial amino acid sequence of SEQ ID NO: 4, that is amino acids8-141; and a polypeptide having an amino acid sequence containing one ormore amino acid modifications in any one of said amino acid sequences ora polypeptide having a homology higher than 51% to any one of said aminoacid sequences and showing an antifungal activity against rice blast.

Purification and isolation of the protein of the present invention canbe accomplished by appropriately combining conventional methods forpurification and isolation of proteins, such as ammonium sulfateprecipitation, ion exchange chromatography (Mono Q, Q Sepharose orDEAE), gel filtration (Superose 6, Superose 12).

For example, ground powder of Pleurotus cornucopiae is extracted with abuffer, and then filtered, and the supernatant is allowed to stand withammonium sulfate at a suitable concentration, e.g. 75% saturation togive precipitates, as described in the examples below. The precipitatesare dialyzed and then eluted by ion exchange chromatography using agradient of salt concentration (e.g., 50 mM-600 mM NaCl), and thenfractions containing a desired protein are recovered on the basis of anantifungal activity. Fractions having a molecular weight of around 30kDa can be recovered by gel filtration. The antifungal protein of thepresent invention has a molecular weight of, but is not limited to,about 15 kDa as determined by SDS-PAGE.

Alternatively, said protein can be obtained in mass quantity byintroducing a DNA sequence consisting of 71 (or 92) to 502 of the DNAsequence of SEQ ID NO: 1 or a DNA sequence consisting of 226 (or 247) to651 of the DNA sequence of SEQ ID NO: 3 into E. coli, yeasts or insectsor certain animal cells by known introduction techniques using anexpression vector capable of amplifying in each host and expressing it.

The amino acid sequence of this protein and the DNA sequence encoding itdisclosed herein can be wholly or partially used to readily isolate agene encoding a protein having a similar physiological activity fromother species, preferably fungi, more preferably Eumycota includingmushrooms, molds and yeasts, and Basidiomycotina including manymushrooms, more preferably mushrooms of Agaricales to which Pleurotuscornucopiae belongs, e.g. Pleurotus ostreatus, Lentinus edodes,Armillariella mellea, Tricholoma matsutake, shimeji mushrooms,Flammulina velutipes, Grifola frondosa, Cantharellus cibarius.,Pleurotus eryngii using basic genetic engineering techniques includinghybridization and PCR. In such cases, these novel proteins are alsoincluded in the scope of the present invention.

Gene for the Antifungal Protein

The present invention also provides a gene encoding the antifungalprotein of the present invention. The type of the gene of the presentinvention is not specifically limited, but may be any of native DNA,recombinant DNA or chemically synthesized DNA, and any genomic DNA cloneor cDNA clone.

The gene of the present invention typically has the nucleotide sequenceshown in SEQ ID NO: 1 or 3. However, the nucleotide sequence of a cloneobtained in the examples shown below is only one example. It iswell-known to those skilled in the art that natural genes includevariations resulting from differences in varieties of the organismsproducing the gene, or from minor mutation depending on differences inecotypes or from minor mutation depending on the presence of closelysimilar isozymes. Accordingly, the gene of the present invention is notlimited to only that having the nucleotide sequence of SEQ ID NO: 1 or 3in the Sequence Listing, and is intended to encompass all genes encodingthe antifungal protein of the present invention.

Especially, the amino acid sequence of the proteins and the DNA sequenceencoding thereof disclosed herein can be wholly or partially used toreadily isolate from other species a gene encoding a protein having asimilar physiological activity using genetic engineering techniques,including hybridization and nucleic acid amplification reactions. Insuch cases, these genes are also included in the scope of the presentinvention.

BLAST searches through GenBank databases using the DNA sequence of agene encoding the Pleurotus cornucopiae-derived antifungal protein (DNAsequence of 71-502 of SEQ ID NO: 1) and the DNA sequence of a geneencoding the second Pleurotus cornucopiae-derived antifungal protein(DNA sequence 226-651 of SEQ ID NO: 3) found only several sequencesshowing homology in a very short range (23 bp) but not the DNA sequenceof streptavidin. This means that the DNA sequence encoding the novelprotein of the present invention is not highly homologous to the DNAsequence of streptavidin on the DNA level.

More specifically, a genetic sequence analysis software programGENETYX-WIN ver 3.2 (Software Development Co., Ltd.) was used to analyzehomology of the total amino acid sequences of the Pleurotus cornucopiaeantifungal proteins of the present invention (tamavidins 1 and 2) tostreptavidin (which differs from streptavidins v2 and v1 by only 9 aminoacids and 1 amino acid, respectively). As a result, the amino acidsequence of tamavidin 1 encoded by tam1 of the present invention showeda homology (amino acid identity) of 46.7% and the amino acid sequence oftamavidin 2 encoded by tam2 showed 48.1%. The homology of the total DNAsequence (SEQ ID NOs: 1 and 3 in the Sequence Listing) to streptavidinwas 53.8% for tam1 and 51.0% for tam2. The homology of the Pleurotuscornucopiae antifungal protein encoded by tam1 to egg white avidin was31.2% in amino acid sequence and 42.4% in DNA sequence, and the homologyof the Pleurotus cornucopiae antifungal protein encoded by tam 2 to eggwhite avidin was 36.2% in amino acid sequence and 41.8% in DNA sequence.The homology between the amino acid sequences of tamavidin 1 andtamavidin 2 and the homology between the DNA sequences of the genes tam1and tam 2 encoding them were 65.5% and 64.5%, respectively.

As compared with streptavidin, tamavidin 1 and tamavidin 2 of thepresent invention are truncated by the N terminal 33 amino acids, butall the tryptophan (W) residues (Gitlin et al. (1988) Biochem. J. 256:pp. 279-282) and tyrosine (Y) residues (Gitlin et al. (1990) Biochem. J.269: pp. 527-530) possibly involved in binding to biotin are conserved,(Y 34 and 45 and W 82, 98 and 110 in the amino acid sequence of SEQ IDNO: 2, Y 34 and 45 and W 80, 96 and 108 in the amino acid sequence ofSEQ ID NO: 4).

The average molecular weights of the regions presumed to be matureprotein regions (stretches 8-143 of the amino acid sequence of SEQ IDNO: 2, and 8-141 of the amino acid sequence of SEQ ID NO: 4) werecalculated at 15158.4 and 14732.2, respectively, close to the averagemolecular weights of mature streptavidin and mature avidin (16490.6 and14342.9, respectively).

Streptavidin is derived from Actinomyces Streptomyces avidinii andavidin is derived from birds (Gallus gallus) egg white. Proteins closelysimilar to streptavidin so far isolated include streptavidins v1 and v2from Streptomyces violaceus (Bayer et al. (1995) Biochim Biophys Acta1263: pp. 60-66), and homologs of the avidin gene so far isolatedinclude an avidin-related gene from avian (avr1-avr5, Keinanen et al.(1994) Eur J Biochem 220: pp. 615-621). Streptavidins v1 and v2 differfrom streptavidin in amino acid sequence by 1 amino acid and 9 aminoacids, respectively, and the avidin-related protein has a homology toavidin of 68-78% in amino acid sequence and 88-92% in DNA sequence. Thehomology between streptavidin and avidin is 29.2% in amino acidsequence, and 46.8% in DNA sequence.

Preferred examples of the antifungal protein of the present invention,tamavidins 1 and 2 are derived from a species of the Basidiomycetes,Pleurotus cornucopiae, and have a homology of 46.7% and 48.1%,respectively, to streptavidin in amino acid sequence and a homology of31.2% and 36.2%, respectively, to avidin in amino acid sequence, asdescribed above. Thus, tamavidins 1, 2 form a third group distinct fromthe streptavidin group of Actinomyces and the avian avidin group. Suchavidin-like protein was first isolated from sources other thanactinomycetes and avian. Tamavidins 1, 2 are avidin-like proteinspresent in mushrooms, and other varieties of mushrooms are likely tocontain similar proteins. The amino acid sequences of tamavidins 1, 2and the DNA sequences of tam1, tam2 can be used to further search andisolate such proteins and genes thereof.

Hybridization conditions used for screening homologous genes are notspecifically limited, but stringent conditions are generally preferred,such as several hours to overnight in 5×SSC, 5×Denhardt's solution, 1%SDS at 25-68° C. as described in Current Protocols in Molecular BiologyVol. 1 (John Wiley and Sons, Inc.) or Molecular Cloning 2nd edition(Sambrook et al. (1989)). The hybridization temperature here is morepreferably 45-68° C. (without formamide) or 30-42° C. (50% formamide).Washing conditions involve e.g. 0.2×SSC at 45-68° C. It is well known tothose skilled in the art that a DNA containing a nucleotide sequencehaving homology higher than a predetermined level can be cloned byappropriately selecting hybridization conditions such as formamidelevel, salt level and temperature, and all of the homologous genes thuscloned are included in the scope of the present invention.

Nucleic acid amplification reactions here include reactions involvingtemperature cycles such as polymerase chain reaction (PCR) (Saiki etal., 1985, Science, 230, pp. 1350-1354), ligase chain reaction (LCR) (Wuet al., 1989, Genomics, 4, pp. 560-569; Barringer et al., 1990, Gene,89, pp. 117-122; Barany et al., 1991, Proc. Natl. Acad. Sci. USA, 88,189-193) and transcription-based amplification (Kwok et al., 1989, Proc.Natl. Acad. Sci. USA, 86, pp. 1173-1177) as well as isothermal reactionssuch as strand displacement amplification (SDA) (Walker et al., 1992,Proc. Natl. Acad. Sci. USA, 89, pp. 392-396; Walker et al., 1992, Nuc.Acids Res., 20, pp. 1691-1696), self-sustained sequence replication(3SR) (Guatelli et. al., 1990, Proc. Natl. Acad. Sci. USA, 87, pp.1874-1878), and Qβ replicase system (Lizardi et al., 1988,BioTechnology, 6, pp. 1197-1202). Other reactions such as nucleic acidsequence-based amplification (NASBA) using competitive amplification ofa target nucleic acid and a mutant sequence disclosed in European PatentNo. 0525882 can also be used. A PCR is preferred.

Homologous genes cloned by hybridization or nucleic acid amplificationreactions as above preferably have a homology of 60% or more, morepreferably 70% or more, still more preferably 80% or more, especially90% or more, most preferably 95% or more to the nucleotide sequenceshown in SEQ ID NO: 1 in the Sequence Listing.

Oligonucleotide

According to the present invention, an oligonucleotide for obtaining anantifungal protein derived from Pleurotus cornucopiae is also provided,which is produced by a method comprising:

selecting two regions from a base sequence of a gene encoding theantifungal protein of SEQ ID NO:1 based on the following requirements;

1) length of each region is 15-30 bases;

2) proportion of G+C content in a base sequence of each region is40-60%;

preparing a single-stranded DNA having a base sequence which isidentical to said region or complementary to said region, or preparingmixture of single-stranded DNAs based on degeneracy of the genetic codewithout changing a sequence of amino acid residues encoded by saidsingle-stranded DNAs; and

optionally preparing a modified version of said single-stranded DNAs,said modification not altering a binding specificity of thesingle-stranded DNAs to the base sequence of the gene encoding saidantifungal protein. The oligonucleotide of the present invention can beused for e.g. hybridization or amplification reactions such as PCR usingsuitable two of the oligonucleotides as a primer pair for detecting orisolating the gene of the present invention.

The oligonucleotide of the present invention preferably has a nucleotidesequence shown in any one of SEQ ID NOs: 10-19 in the Sequence Listing.The nucleotide sequences of SEQ ID NOs: 10-13 were designed on the basisof the amino acid sequence of SEQ ID NO: 9 as PCR primers for cloning agene fragment encoding a part of the protein and comprise all the basescapable of encoding the amino acid. The nucleotides of SEQ ID NOs: 14-17are primers synthesized for primer walking for decoding the totalnucleotide sequences of tam1 and tam2 genes. The nucleotides of SEQ IDNOs: 18-19 are PCR primers prepared on the basis of SEQ ID NO: 3 foramplifying the total ORF to construct an expression vector forexpressing recombinant tamavidin 2 protein having the amino acidsequence of SEQ ID NO: 4.

A partial fragment of the gene of the present invention can be isolatedby nucleic acid amplification reactions such as PCR using a cDNA libraryof Pleurotus cornucopiae fruit body as a template with an appropriatepair of the above oligonucleotides. A full-length cDNA clone can beisolated by screening the cDNA library with an amplification productthus obtained as a probe by e.g. plaque hybridization. The proceduresand conditions for nucleic acid amplification reactions and the plaquehybridization conditions are well-known to those skilled in the art.

For example, hybridization conditions of rather low stringency may beused, such as, but not limited to, room temperature and washing athigher salt concentrations such as 2×SSC at 37° C. as described inCurrent Protocols in Molecular Biology Vol. 1 (John Wiley and Sons,Inc.) or Molecular Cloning (Sambrook et al., supra.).

Preparation of Recombinant Antifungal Proteins

The protein of the present invention has a very strong antifungalactivity. For example, it completely inhibits the germination of sporesof rice blast (M. grisea) at a concentration as low as 50 ng/ml (seeExample 4 below). No germination of spores appears at this concentrationeven after extended incubation, suggesting that the effect of theprotein of the present invention against rice blast may be afungus-killing effect rather than partial inhibition of growth. To ourknowledge, no antifungal proteins that can completely inhibit the growthof pathogenic microorganism at such a low concentration (on the order ofnanograms) have been reported thus far. In the examples below, a majorrice pathogen, rice blast, was used as a plant pathogen for theantifungal assay for purifying an antifungal protein, but it is highlypossible that the Pleurotus cornucopiae antifungal proteins identifiedherein have comparable antifungal effects against other plant pathogenicdamage such as Rhizoctonia solani.

Thus, the Pleurotus cornucopiae-derived antifungal protein of thepresent invention has a potent antifungal activity, so that it can beused in formulations such as antifungal agents and pesticides, which cancontain the antifungal protein in an active form. In this case, thepresent protein is purified from Pleurotus cornucopiae by using e.g. anion exchange column or a gel filtration column as described in theexamples below. However, the Pleurotus cornucopiae antifungal protein ofthe present invention can be prepared more conveniently in mass quantityby introducing and expressing DNA having the nucleotide sequence of71-502 of SEQ ID NO: 1 or 226-651 of SEQ ID NO: 3 encoding the proteinin E. coli, yeasts, insects or animal cells using an expression vectorcapable of amplifying in each host (Example 5).

The present invention also provides a recombinant vector containing thegene of the present invention. Methods for inserting a DNA fragment ofthe gene of the present invention into a vector such as a plasmid aredescribed in e.g. Sambrook, J. et al, Molecular Cloning, A LaboratoryManual (2nd edition), Cold Spring Harbor Laboratory, 1.53 (1989).Commercially available ligation kits (e.g. available from TAKARA SHUZOCO., LTD.) can be conveniently used. Thus obtained recombinant vectors(e.g. recombinant plasmids) are introduced into host cells (e.g., E-coilTB1, LE392 or XL-1Blue).

Suitable methods for introducing a plasmid into a host cell include theuse of calcium phosphate or calcium chloride/rubidium chloride,electroporation, electroinjection, chemical treatment with PEG or thelike, the use of a gene gun described in Sambrook, J. et al., MolecularCloning, A Laboratory Manual (2nd edition), Cold Spring HarborLaboratory, 1.74 (1989).

Vectors can be conveniently prepared by linking a desired gene by astandard method to a recombination vector available in the art (e.g.plasmid DNA). Specific examples of suitable vectors include, but are notlimited to, E. coli-derived plasmids such as pBluescript, pUC18, pUC19,pBR322, pTrc99A.

Expression vectors are especially useful for the purpose of producing adesired protein. The types of expression vectors are not specificallylimited so far as they can express a desired gene in various prokaryoticand/or eukaryotic host cells to produce a desired protein, butpreferably include expression vectors for E. coli such as pQE-30,pQE-60, pMAL-C2, pMAL-p2, pSE420; expression vectors for yeasts such aspYES2 (genus Saccharomyces), pPIC3.5K, pPIC9K, pAO815 (all genusPichia); and expression vectors for insects such as pBacPAK8/9, pBK283,pVL1392, pBlueBac4.5.

Transformants can be prepared by introducing a desired expression vectorinto a host cell. Suitable host cells are not specifically limited sofar as they are compatible with expression vectors and transformable,and include various cells such as natural cells or artificiallyestablished recombinant cells commonly used in the field of the presentinvention. Examples are bacteria (Escherichia, Bacillus), yeasts(Saccharomyces, Pichia), animal cells, insect cells, plant cells, etc.

Host cells are preferably E. coli, yeasts or insect cells, specificallyE. coli such as M15, JM109, BL21; yeasts such as INVSc1 (genusSaccharomyces), GS115, KM71 (all genus Pichia); insect cells such asBmN4, silkworm larvae. Examples of animal cells are those derived frommouse, Xenopus, rat, hamster, simian or human or culture cell linesestablished from these cells. Plant cells include those derived fromtobacco, Arabidopsis, rice, maize, wheat, etc., but are not specificallylimited so far as they can be cultured.

When a bacterium, especially E. coli is used as a host cell, theexpression vector generally consists of at least a promoter/operatorregion, an initiation codon, a gene encoding a desired antifungalprotein, a termination codon, a terminator and a replicable unit.

When a yeast, plant cell, animal cell or insect cell is used as a hostcell, the expression vector generally preferably contains at least apromoter, an initiation codon, a gene encoding a desired antifungalprotein, a termination codon and a terminator. It may also contain a DNAencoding a signal peptide, an enhancer sequence, non-translated 5′ and3′ regions of the desired gene, a selectable marker or a replicableunit, etc., if desired.

A preferred initiation codon in vectors of the present invention is amethionine codon (ATG). Termination codons may be conventionaltermination codons (for example, TAG, TGA, TAA).

The replicable unit means a DNA capable of replicating the entire DNAsequence in a host cell, and includes natural plasmids, artificiallymodified plasmids (plasmids prepared from natural plasmids) andsynthetic plasmids, etc. Preferred plasmids are pQE30, pET or pCAL ortheir artificial modifications (DNA fragments obtained by treatingpQE30, pET or pCAL with suitable restriction endonucleases) for E. coli;pYES2 or pPIC9K for yeasts; and pBacPAK8/9 for insect cells.

Enhancer sequences and terminator sequences may be those commonly usedby those skilled in the art such as those derived from SV40.

As for selectable markers, those commonly used can be used by standardmethods. Examples are genes, which provide resistance to antibioticssuch as tetracycline, ampicillin, kanamycin, neomycin, hygromycin orspectinomycin.

Expression vectors can be prepared by consecutively and cyclicallylinking at least the above-described promoter, initiation codon, geneencoding a desired antifungal protein, termination codon and terminatorregion to a suitable replicable unit. In this process, a suitable DNAfragment (such as a linker or another restriction enzyme site) can beused by standard methods such as digestion with a restriction enzyme orligation with T4DNA ligase, if desired.

Introduction [transformation (transduction)] of expression vectors ofthe present invention into host cells can be conducted by using knowntechniques.

For example, bacteria (such as E. coli, Bacillus subtilis) can betransformed by the method of Cohen et al. [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], the protoplast method [Mol. Gen. Genet., 168, 111(1979)] or the competent method [J. Mol. Biol., 56, 209 (1971)];Saccharomyces cerevisiae can be transformed by the method of Hinnen etal [Proc. Natl. Acad. Sci. USA, 75, 1927 (1978)] or the lithium method[J. Bacteriol., 153, 163 (1983)]; plant cells can be transformed by theleaf disc method [Science, 227, 129 (1985)] or electroporation [Nature,319, 791 (1986)]; animal cells can be transformed by the method ofGraham [Virology, 52, 456 (1973)]; and insect cells can be transformedby the method of Summers et al. [Mol. Cell. Biol., 3, 2156-2165 (1983)].

Plant transforming vectors are especially useful for the purpose ofcreating a disease-resistant plant using a DNA fragment of the presentinvention. The types of vectors for plants are not specifically limitedso far as they can express the gene of interest in plant cells toproduce the protein, but preferably include pBI221, pBI121 (Clontech),and vectors derived there from. Especially, examples of vectors fortransforming monocotyledons include pIG121Hm and pTOK233 (Hiei et al.,Plant J., 6,271-282 (1994)), and pSB424 (Komari et al., Plant J.,10,165-174 (1996)).

Transgenic plants can be prepared by replacing the β-glucuronidase (GUS)gene in the above vectors with a DNA fragment of the present inventionto construct a plant-transforming vector and introducing it into aplant. The plant-transforming vector preferably contains at least apromoter, an initiation codon, a desired gene (a DNA sequence of thepresent invention or a part thereof), a termination codon and aterminator. It may also contain a DNA encoding a signal peptide, anenhancer sequence, non-translated 5′ and 3′ regions of the desired gene,a selectable marker region, etc., if desired.

Promoters and terminators are not specifically limited so far as theyare functional in plant cells, among which constitutive expressionpromoters include the 35S promoter initially being inserted in the abovevectors as well as promoters for actin and ubiquitin genes. However, aninducible promoter may be more preferably inserted. This allowstransgenic plants to be resistant to a pest by producing the proteinonly when they come into contact with it. Suitable inducible promotersinclude promoters of genes of phenylalanine ammonia-lyase, chitinase,glucanase, thionine, and osmosin and other promoters of genes respondingto pests or stresses.

Methods for the gene transduction into a plant include the use ofAgrobacterium (Horsch et al., Science, 227, 129 (1985); Hiei et al.,Plant J., 6, pp. 271-282 (1994)), electroporation (Fromm et al., Nature,319, 791 (1986)), PEG (Paszkowski et al., EMBO J., 3, 2717 (1984)),microinjection (Crossway et al., Mol. Gen. Genet., 202, 179 (1986)),particle bombardment (McCabe et al., Bio/Technology, 6, 923 (1988)), butare not specifically limited so far as they are suitable fortransfecting a gene into a desired plant. The species of host plants arenot specifically limited, either, so far as they are compatible with theplant transforming vectors of the present invention and transformable,specifically plants commonly used in the field of the present invention,e.g. dicotyledons such as tobacco, Arabidopsis, tomato, cucumber,carrot, soybean, potato, beet, turnip, Chinese cabbage, rape, cotton andpetunia; and monocotyledons such as rice, corn and wheat.

The protein of the present invention can be expressed (produced) byculturing transformed cells containing an expression vector prepared asdescribed above in a nutrient medium. The nutrient medium preferablycontains a carbon, inorganic nitrogen or organic nitrogen sourcenecessary for the growth of host cells (transformants). Examples ofcarbon sources include e.g. glucose, dextran, soluble starch, sucroseand methanol. Examples of inorganic or organic nitrogen sources includeammonium salts, nitrates, amino acids, corn steep liquor, peptone,casein, beef extract, soybean meal and potato extract. If desired, othernutrients (e.g. inorganic salts such as sodium chloride, calciumchloride, sodium dihydrogen phosphate and magnesium chloride; vitamins;antibiotics such as tetracycline, neomycin, ampicillin and kanamycin)may be contained.

Incubation takes place by techniques known in the art. Incubationconditions such as temperature, the pH of the medium and the incubationperiod are appropriately selected to produce the protein of the presentinvention in mass quantity. For expression in E. coli, incubationconditions for expressing a recombinant protein include, but are notlimited to, incubation at a temperature of 4-40° C. and induction with0.01-5.0 mM IPTG.

The protein of the present invention can be obtained from the culturesas follows. When the protein of the present invention accumulates inhost cells, the host cells are collected by centrifugation or filtrationor the like and suspended in a suitable buffer (e.g. a buffer such asabout 10 M-100 mM Tris buffer, phosphate buffer, HEPES buffer or MESbuffer at a pH depending on the buffer used, but desirably in the rangeof pH 5.0-9.0), then the cells are disrupted by a method suitable forthe host cells used and centrifuged to collect the contents of the hostcells. When the protein of the present invention is secreted outsidehost cells, the host cells and the culture medium are separated bycentrifugation or filtration or the like to give a culture filtrate. Thehost cell lysates or the culture filtrates can be used to isolate/purifythe protein of the present invention directly or after ammonium sulfateprecipitation and dialysis.

An isolation/purification method is as follows. When the protein ofinterest is tagged with 6× histidine, GST, maltose-binding protein orthe like, conventional methods based on affinity chromatography suitablefor each tag can be used. As a non-limiting example, a recombinantantifungal protein tagged with 6× histidine at the N-terminus wasexpressed in Example 4 below. This recombinant protein was purifiedusing Ni-NTA agarose (Qiagen) having affinity for 6× histidine. When theprotein of the present invention is produced without using these tags,the method described in detail in the examples below based on ionexchange chromatography can be used, for example. These methods may becombined with gel filtration or hydrophobic chromatography, isoelectricchromatography or the like. Purification on an iminobiotin affinitycolumn can also be applied as described by Hofmann et al., (Proc. Natl.Acad. Sci. USA, 77: pp. 4666-4668 (1980)). In Example 5 below, therecombinant protein, tamavidin 2 was obtained at a yield of 1 mg from 50mL of E. coli cultures.

The antifungal proteins of the present invention obtained by geneticengineering techniques or purified from natural sources as describedabove have antifungal activity. The antifungal activity can bedetermined by, but not limited to, incubating microtiter platescontaining spores of rice blast suspended in a culture medium (e.g. ½PD, sucrose-peptone) in the presence of the antifungal protein of thepresent invention at a predetermined concentration, e.g. 10 ng/ml-1000ng/ml, preferably 50 ng/ml at 28° C. for 48 hours, and evaluatingwhether or not the growth/proliferation of rice blast (e.g. extension ofhyphae) is inhibited as compared with a control not containing theantifungal protein (Example 4).

Alternatively, the following assay can also be applied. A colony of riceblast is placed at the center of an agar medium prepared in a Petri dishand a predetermined amount of an aqueous solution of the antifungalprotein of the present invention is dropped around the colony, and thePetri dish is incubated at 28° C. for about 48 hours to a week. Then,the antifungal activity can be assayed by evaluating whether or not theextension of hyphae of rice blast in regions treated with the antifungalprotein is inhibited as compared with untreated regions.

Antifungal Agents

The proteins of the present invention have a potent antifungal activity.For example, it inhibits the growth of hyphae of rice blast at a lowconcentration such as 50 ng/ml in our antifungal assay. In the examplesbelow, M. grisea and Rhizoctonia solani, which cause extensive damage torice crops, were used as plant pathogens for the antifungal assay. ThePleurotus cornucopiae antifungal protein identified herein showed anantifungal effect against them. The protein of the present invention ismost likely to have an antifungal effect against plant pathogenicmicroorganisms other than M. grisea.

Thus, the Pleurotus cornucopiae-derived antifungal protein of thepresent invention has a potent antifungal activity, so that it can beused in formulations such as antifungal agents and pesticides, which cancontain the antifungal protein in an active form. In this case, theprotein of the present invention can be prepared in mass quantity byinserting a DNA sequence encoding the protein of the present inventioninto an expression vector functional in e.g. E. coli or yeasts asdescribed above.

The antifungal protein of the present invention is a novelstreptavidin-like protein, suggesting that it bind to one of vitamins,biotin (vitamin H). Rice blast is known to require biotin for itsgrowth. These facts suggest that the present antifungal protein binds tofree biotin present in assay media to induce biotin deficiency in themedia, with the result that the growth of rice blast was inhibited. Infact, the antifungal activity of tamavidin 1 of the present inventionwas abolished when biotin was excessively added into the assay medium asdescribed in Example 4 below. We further found that commerciallyavailable streptavidin and avidin also have an antifungal effect againstrice blast similar to tamavidin 1, and demonstrated that this effect isalso abolished by biotin.

The present invention suggested the possibility that resistance todisease, especially to rice blast can be conferred on plants bycontrolling the amount of one of vitamins, biotin. The possibility thatdisease-resistance can be conferred by controlling a vitamin has notbeen so far known. This is a novel concept. This concept is alsoincluded in the present invention. For example, a formulation containingthe antifungal protein of the present invention as an active ingredientcan be used as a pesticide. In this case, biotin-binding proteins otherthan the antifungal protein of the present invention (e.g. streptavidinof Streptomyces avidinii and egg white avidin, and homologs thereof) arealso included in the same concept.

Thus, the present invention provides an antifungal agent containing theantifungal protein of the present invention as an active ingredient.Normally, the antifungal agent of the present invention can besystemically or locally applied to plants.

Dispersion dose of the antifungal agent depends on the type of plant,growth stage, condition, dispersion method, treating time, the type ofthe protein applied (e.g. a full-length protein or a protein obtained bysubstitution, deletion, insertion and/or addition of a part of theformer protein), the weather and the soil of the site where the plantgrows, and other factors, and the antifungal agent can be dispersed onceor more daily or at intervals of several days. The antifungal agent ofthe present invention can also be dispersed in admixture withsolubilizers, suspending agents, emulsifiers, etc., if necessary.Aqueous or non-aueous solubilizers and suspending agents are mixed as atleast one inert diluent with one or more active substances. Examples ofaqueous diluents include distilled water and saline. Examples ofnon-aqueous diluents include propylene glycol, polyethylene glycol, andvegetable oils such as olive oil and alcohols such as ethanol.

Such antifungal compositions may further contain auxiliary agents suchas preservatives, humectants, emulsifiers, dispersants or stabilizers(e.g. arginine, aspartic acid, etc.).

These compositions are sterilized by filtration through a bacteriostaticfiler or the addition of a bactericide or irradiation, if necessary.They can also be prepared as sterile solid compositions by, for example,freeze-drying and then dissolved in distilled water or other solventsbefore use.

The dosage form of the antifungal agent thus obtained may beappropriately determined depending on the purpose, i.e. it can beapplied in the form of tablets, pills, dusts, granules, solutions,emulsions, etc. in admixture with the additives mentioned above.

Disease-resistant plants can also be created by inserting a geneencoding the antifungal protein of the present invention into a plant.Thus, a disease-resistant plant can be created by e.g. introducing aplant with a construct in which a promoter functional in the plant islinked to a gene encoding the antifungal protein of the presentinvention and a terminator functional in the plant is further addeddownstream. In this case, a DNA sequence encoding a signal peptide forextracellular secretion functional in the plant may be added to the 5′side of the gene encoding the antifungal protein of the presentinvention in order to promote the secretion of tamavidin outside plantcells. Alternatively, the codon usage of the gene can be adapted formonocotyledons or dicotyledons without affecting the amino acids topromote accumulation of the antifungal protein inside or outside plantcells. Methods for creating disease-resistant plants using combinationsof these means are also included in the present invention.

Applications of tamavidin, avidin, streptavidin or closely similarproteins thereto for creating disease-resistant plants and to plantsother than rice are also included in the present invention. Although thepathogenic fungus analyzed herein is rice blast, it is quite possiblethat other plant pathogenic fungi and pathogenic bacteria requiringbiotin for their growth are also covered.

Moreover, similar effects may naturally be produced against not onlyplant pathogenic microorganisms but also animal pathogenic fungiessentially requiring biotin for their growth, especially pathogenicmicroorganisms to human and domestic animals, and therefore, the presentinvention encompasses the uses of the antifungal protein of the presentinvention, avidin or streptavidin, and closely similar proteins theretoas therapeutic agents in such scenes.

The DNA sequence of streptavidin has already been disclosed (Garwin etal., WO/8602077), but the DNA sequences of tam1 and tam2 of the presentinvention were not matched to the DNA of streptavidin during ordinarydatabase searches and actually showed homology to the DNA ofstreptavidin at only 51.0-53.8% during forced comparison using a nucleicacid/amino acid sequence analysis software program, as described above.

Streptavidin and avidin have already been widely used as experimentalreagents in various scenes in molecular biology, biochemistry or thelike because they have very strong binding affinity to biotin andderivatives thereof. For example, they are used in detection systems ofnucleic acids and proteins (Liang. WO/9707244) or purification methodsbased on the binding affinity to biotin of streptavidin or avidinexpressed as a fusion protein (Skerra et al. EP835934, Kopetzki.WO/9711186). Tamavidin 1 and tamavidin 2 of the present invention canalso be used in these applications currently widely known or reported.

Plant-related applications of streptavidin or avidin so far reportedinclude the creation of male sterile plants using avidin (Howard andAlbertsen. WO/9640949), the application of streptavidin or avidin asinsecticidal protein (Czapla et al. WO/9400992), and the production ofavidin in plants (Baszczynski et al. U.S. Pat. No. 5,767,379). The usesof streptavidin or avidin described in these documents can also apply tothe Pleurotus cornucopiae-derived antifungal protein of the presentinvention.

REFERENCES

-   1. Schlumbaum et al. (1986) Nature 324: pp. 365-367-   2. Mauch et al. (1988) Plant Physiol. 88: pp. 936-942-   3. Japanese Domestic announcement No. 505048/96-   4. Oita et al. (1996) Biosci. Biotech. Biochem. 60: pp. 481-483-   5. Terras et al. (1992) J. Biol. Chem. 267: pp. 15301-15309-   6. Japanese Domestic announcement No. 501424/97-   7. Broglie et al. (1991) Science 254: pp. 1194-1197-   8. Terras et al. (1995) The Plant Cell 7: pp. 573-588-   9. Nishizawa et al. (1999) Theor Appl Genet. 99:383-390-   10. Alexander et al. (1993) Proc. Natl. Acad. Sci. USA 90: pp.    7327-7331-   11. Wu et al. (1995) Plant Cell 7: pp. 1357-1368-   12. Hain et al. (1993) Nature 361: pp. 153-156-   13. Bayer et al. (1995) Biochim Biophys Acta 1263: pp. 60-66-   14. Argarana et al. (1986) Nucleic Acids Res 14: pp. 1871-1882-   15. Freitag et al. (1997) Protein Sci. 6: pp. 1157-1166-   16. Gope et al. (1987) Nucleic Acids Res 15: pp. 3595-3606-   17. Keinanen et al. (1994) Eur J Biochem 220: pp. 615-621-   18. Gitlin et al. (1988) Biochem. J 256: pp. 279-282-   19. Gitlin et al. (1990) Biochem J 269: pp. 527-530-   20. Hofmann et al., Proc. Natl. Acad. Sci. USA, 77: pp. 4666-4668    (1980)-   21. Garwin et al. WO/8602077-   22. Liang. WO/9707244-   23. Skerra et al. EP835934-   24. Kopetzki. WO/9711186-   25. Howard and Albertsen. WO/9640949-   26. Czapla et al. WO/9400992-   27. Baszczynski et al. U.S. Pat. No. 5,767,379.

The following examples further illustrate the present invention without,however, limiting the invention thereto.

EXAMPLES Example 1 Construction of an Assay System 1) Establishment ofan Assay System

Cultivation of pathogenic fungi: Magnaporthe grisea (rice blast) (race337, strain TUS-1 obtained from National Agricultural Research Centerfor Tohoku Region of the Ministry of Agriculture, Forestry and Fisheriesof Japan) was cultured on an oatmeal medium (Difco, supplemented with 1%sucrose) to give conidia for use as an inoculum. The spores were storedat −80° C. in 10% glycerol, if necessary.

Rhizoctonia solani (strain JT872) was cultured on ½ potato dextrosebroth (PD, Difco) for 2 days, and three mycelia of about 5 mm weregently ground in ½ PD in a Teflon homogenizer to give hyphal fragmentsfor use as an inoculum.

These inocula were added to 96-well microtiter plates (Corning) at adensity of about 1,000 conidia of M. grisea per well or about 300 hyphalfragments of R. solani per well in 100 μl of ½ PD and incubated in anincubator at 28° C. for 48 hours. The growth of the fungi was monitoredby measuring the absorbance at 595 nm with a microplate reader(Benchmark, Bio-Rad).

2) Extraction of Protein from Pleurotus cornucopiae

After 100 g of commercially available fruit bodies of Pleurotuscornucopiae were finely cut with scissors in advance, they were frozenin liquid nitrogen and ground in a mortar into fine powder, and thenextracted with 300 ml of 100 mM HEPES-KOH buffer, pH 7.5 at 4° C. for 30minutes with gentle stirring. The extract was filtered through Miraclothand then centrifuged at 10,000×g for 20 minutes. Then, the supernatantwas allowed to stand at 4° C. overnight with 75% saturation ammoniumsulfate. Then, precipitates were obtained by centrifugation at 15,000×gfor 20 minutes and dissolved in 3 ml of 10 mM HEPES-KOH buffer, pH 7.5and dialyzed against 20 mM HEPES-KOH buffer, pH 7.5 using a dialysistube (Spectra/Por1 MWCO 6-8000, Spectrum Medical Industries). Insolubleswere removed by centrifugation to give a Pleurotus cornucopia proteinsample. The protein level of the Pleurotus cornucopia protein sample wasdetermined by the Bradford method using bovine serum albumin (BSA) as astandard protein.

Example 2 Purification of Antifungal Protein

1) Antifungal Activity of the Crude Pleurotus cornucopiae Protein Sample

The culture systems of M. grisea and R. solani were added with a givenamount the crude P. cornucopiae protein sample added immediately afterstarting cultivation, incubated for 2 days (46-48 hours), and thenevaluated for an antifungal activity by measuring the absorbance. Theresults showed that the Pleurotus cornucopiae extract contained asubstance having a high antifungal activity against both Magnaporthegrisea and Rhizoctonia solani. Complete inhibition of germination andinhibition of the growth of hyphae were observed against M. grisea andinhibition of the growth of hyphae was observed against R. solani. Asfor cells of M. grisea, the cytoplasm was separated from the cell walland looked like plasmolysis.

To further analyze the nature of the antifungal activity detected,residual activity was tested after heating. The antifungal assay wasperformed after heating at 60 and 80° C. for 10 minutes. The strength ofactivity was estimated by diluting the protein sample. As a result, theantifungal activity against both M. grisea and R. solani was comparablebefore and after heating at 60° C. However, the antifungal activityagainst R. solani disappeared after heating at 80° C. In contrast, a newactivity was shown against M. grisea by swelling hyphal apices to stopthe growth after heating at 80° C., though the activity of inducingplasmolysis was lost (FIG. 1).

To know the approximate molecular weight of the core substance governingthese activities contained in heated fractions of the crude Pleurotuscornucopiae protein sample, the sample was fractionated through anultrafiltration membrane to study an antifungal activity by separatingthe sample into fractions which passed through or not an UltrafreeMC10,000 NMWL filter unit (cut-off molecular weight 10000, Millipore)used as an ultrafiltration membrane. As a result, all the activitiesexisted in only fractions retained on the membrane. Thus, the molecularweight of the active core was estimated to be at least 10000 or higher.

2) Purification by Ion Exchange Chromatography

Then, the antifungal protein was purified. Initially, 150 mg/20 ml ofthe crude protein sample was loaded on an home-built column (innerdiameter 1.5 cm×height 10 cm, column volume 10 ml) packed with an ionexchanger Q Sepharose FF (Pharmacia) to partially purify the antifungalprotein. A buffer of 50 mM Tris pH 8.0, 50 mM NaCl to 50 mM Tris pH 8.0,600 mM NaCl as elution buffer with a gradient (50 mM to 600 mM NaCl) wasused at a flow rate of 2 ml/min over 100 minutes. A part of eachfraction (12.5 ml) was subjected to the antifungal assay against M.grisea and SDS-PAGE electrophoresis. The protein solution of eachfraction was reacted with an equivalent amount of 2×SDS running buffer(Sambrook et al. (1989) Molecular Cloning 2nd edition, Cold SpringHarbor) at 95° C. for 5 minutes, and then run by SDS-PAGEelectrophoresis according to the method of Laemmli (Laemmli (1970)Nature 227: pp. 680-685.). The gel used is 15% PAGEL (ATTO) and theprotein was detected with a Silver Stain II kit Wako (Wako Pure ChemicalIndustries). To estimate the approximate molecular weight and the amountof the protein, a molecular weight marker was run (LMW marker kit:Pharmacia LKB, sizes 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, 14.4kDa). The electrophoretic patterns of protein by silver staining areshown in FIG. 2 in relation to the strength of an antifungal activity.

Two peaks appeared as antifungal activities at NaCl concentrations of160 mM and 240 mM-280 mM. The protein contained in the peak at 160 mMacted by swelling hyphal apices to stop the growth, and did notdisappear after heating at 70° C. for 10 minutes. However, the proteincontained in the peat at 240 mM-280 mM induced plasmolysis in M. griseaand disappeared after heating at the same temperature. Accordingly, anattempt was made to purify the antifungal protein contained in the peatat 160 mM.

The fractions corresponding to the NaCl concentration of 120 mM-240 mMwere transferred to a dialysis tube (Spectra/Por1 MWCO 6-8000, SpectrumMedical Industries), and dialyzed against 50 mM Tris-HCl pH 8.0, 50 mMNaCl at 4° C. overnight. Concentration on Centriprep-10 (cut-offmolecular weight 10,000, Amicon) was followed by heating at 70° C. for30 minutes. After centrifugation, the supernatant was filtered through a0.22 μm filter. This protein sample (about 10 ml) was loaded on MonoQ HR5/5 (Pharmacia) to separate/purify antifungal protein. A buffer of 50 mMTris-HCl, pH 8.0, 50 mM NaCl to 50 mM Tris-HCl, pH 8.0, 500 mM NaCl wasused as elution buffer with a gradient (50 mM to 500 mM NaCl) at a flowrate of 1 ml/min over 40 minutes starting at 20 minutes after loadingthe sample. A part of each fraction (1 ml) was subjected to theantifungal assay against M. grisea and SDS-PAGE electrophoresis.

The HPLC chart is shown in FIG. 3 in relation to the strength of anantifungal activity. The results show that an elution peak of theantifungal protein appeared around an ionic strength (NaClconcentration) of 200 mM-260 mM.

The electrophoretic pattern is shown in FIG. 4 in relation to thestrength of an antifungal activity. The figure at the top of each lanecorresponds to the fraction number in FIG. 3. Careful examination ofprotein bands possibly related to an antifungal activity found two bandsof about 15 kD as likely candidates (arrows in FIG. 4). The strength ofthe bands and the level of an antifungal activity are positivelycorrelated, suggesting the possibility that the bands may be the coreantifungal protein.

3) Purification by Gel Filtration and Estimation of the Molecular Weight

To purify Pleurotus cornucopiae antifungal protein and estimate thenative molecular weight, Mono Q fractions #41-46 obtained as above wereconcentrated on an Ultrafree MC10,000 NMWL filter unit (Millipore) andloaded on a gel filtration column Superose 6 HR 10/30 (Pharmacia). Thebuffer used is 50 mM MES—NaOH pH 6.0, 50 mM NaCl at a flow rate of 0.5ml/min. The molecular weights and the approximate elution times of theprotein were predicted by Gel filtration standard (BIO-RAD), and thenMonoQ fractions having an antifungal activity were loaded.

As a result, a sharp peak appeared at 30 kDa when the protein wasmonitored at A280 (FIG. 5). The Antifungal activity was concentrated atthe peak and close to 30 kDa. This shows that the antifungal activity ofPleurotus cornucopiae is derived from a single protein having amolecular weight of about 30 kDa as determined by gel filtration. Wheneach fraction (0.25 ml) was silver-stained after SDS-PAGE, a band of 15kDa shown in FIG. 4 was detected only around 30 kDa (FIG. 6). No bandother than 15 kDa appeared, strongly suggesting again that the proteinof 15 kDa contributes to an antifungal activity. The amount of theantifungal protein was estimated from a molecular weight marker (trypsininhibitor at 20.1 kDa) by a densitometer and the 50% growth inhibitionconcentration against M. grisea was calculated at about 50 ng/ml. Theamount of the antifungal protein that can be purified from a crudeweight of 100 g of Pleurotus cornucopiae fruit bodies by the abovemethod was about 0.2 mg.

Example 3 Isolation of cDNA

1) Determination of the Amino Acid Sequence of Pleurotus cornucopiaeAntifungal Protein

The Superose 6 fraction obtained as above was concentrated on anUltrafree MC 10,000 NMWL (Millipore) and subjected to SDS-PAGEelectrophoresis. The fraction was transferred to a PVDF membrane(Millipore) in a buffer system containing neither Tris nor glycine, andlightly stained with Coomassie Brilliant Blue R-250 and then destained.Then, the protein band of 15 kDa possibly contributing to an antifungalactivity was excised. The protein of 15 kDa was partially digested withlysyl endopeptidase (Wako Pure Chemical Industries) or V8 protease (WakoPure Chemical Industries).

As a result, a 14 kDa fraction was obtained by digestion with lysylendopeptidase and 14 kDa and 12 kDa fractions were obtained by digestionwith V8 protease. These bands were also transferred after migration.Then, the N-terminal amino acid sequence was determined by Edmandegradation using a gas-phase protein the Sequencer (HPG1005A ProteinSequencing System).

As a result, the following 44 amino acids were determined from the 15kDa protein:

(SEQ ID NO: 5) N′-Leu Xaa Gly Xaa Trp Tyr Asn Glu Leu Gly Xaa Xaa Met Asn Leu Thr Ala Asn Lys Asp Gly Ser Leu Xaa Gly Thr Tyr His Ser Asn Val Gly Glu Val Pro Xaa Xaa Tyr His Leu Ala Gly Arg  Tyr-C′wherein and hereinafter Xaa is unknown. The following 50 amino acidswere determined from the lysyl endopeptidase digest of the 14 kDaprotein:

(SEQ ID NO: 6) N′-Asp Gly Ser Leu Thr Gly Thr Tyr His Ser Asn Val Gly Glu Val Pro Pro Thr Tyr His Leu Ser Gly Arg Tyr Asn Leu Gln Pro Pro Ser Gly Gln Gly Val Thr Leu Gly Xaa Ala Val Ser Phe Glu Asn Thr Xaa Ala Asn Val-C′.The following 21 amino acids were determined from the V8 protease digestof the 14 kDa protein:

(SEQ ID NO: 7) N′-Leu Thr Gly Thr Trp Tyr Asn Glu Leu Gly Ser Thr Met Asn Leu Thr Ala Asn Lys Asp  Gly-C′.The following 23 amino acids were determined from the 12 kDa protein:

(SEQ ID NO: 8) N′-Leu Thr Gly Thr Xaa Tyr Asn Glu Leu Gly Ser Thr Xaa Asn Leu Thr Ala Asn Xaa Asp Gly  Xaa Leu-C′.

Finally, the following 69 amino acids were determined:

(SEQ ID NO: 9) N′-Leu Thr Gly Thr Trp Tyr Asn Glu Leu Gly Ser Thr Met Asn Leu Thr Ala Asn Lys Asp Gly Ser Leu Thr Gly Thr Tyr His Ser Asn Val Gly Glu Val Pro Pro Thr Tyr His Leu Ser Gly Arg Tyr Asn Leu Gln Pro Pro Ser Gly Gln Gly Val Thr Leu Gly Xaa Ala Val Ser Phe Glu Asn Thr Xaa Ala Asn Val-C′.

2) Design of Degenerate Primers

Based on the amino acid sequence determined in 1), four primerscontaining all the possible bases were synthesized. The figures inparentheses show the degree of degeneracy.

(SEQ ID NO: 10) TMR1: 5′-acnggnacntggtayaayg-3′ (256)(corresponding to the amino acid residues Thr2 to Glu8 of SEQ ID NO: 9)(SEQ ID NO: 11) TMR2: 5′-garytiggiwsnacnatgaa-3′ (256)(corresponding to the amino acid residues Glu8 to Asn14 of SEQ ID NO: 9)(SEQ ID NO: 12) TMF1: 5′-gtrttytcraaiswiacn-3′ (128)(corresponding to the amino acid residues A1a59to Thr65 of SEQ ID NO: 9) (SEQ ID NO: 13) TMF2:5′-cciarigtnacnccytgncc-3′ (256)(corresponding to the amino acid residues G1y51to G1y57 of SEQ ID NO: 9)wherein “i” means “inosine”, “r” means “g or a”, “y” means “c or t”, “w”means “a or t”, “s means “g or c”, and “n” means “a or g or c or t”,respectively.3) Construction of a cDNA Library of Pleurotus cornucopiae Fruit Body

Total nucleic acid was extracted from Pleurotus cornucopiae fruit bodyby the SDS phenol method and total RNA was recovered by lithium chlorideprecipitation. Then, Pleurotus cornucopiae mRNA was prepared from thetotal RNA using an mRNA purification kit (Pharmacia). From about 5 g offruit bodies, 10 μg of mRNA was obtained, of which 5 μg was used in aZAP cDNA synthesis kit (Stratagene) to synthesize cDNA. About 0.5-5 kbof cDNA was fractionated by gel filtration and ligated to a Uni-ZAP XRvector (Stratagene) and packaged with Gigapack III (Stratagene). All theprocedures were carried out as according to instructions in the kit. Thetiter of thus constructed cDNA library of Pleurotus cornucopiae fruitbody was estimated at about 3,000,000 pfu.

4) Preparation of Probes by RT-PCR

PCR was performed using the primers synthesized in 2) and the cDNAsynthesized in 3) as a template to try to amplify a partial length cDNAof Pleurotus cornucopiae antifungal protein suitable as a probe forscreening libraries. The reaction conditions were as follows: 50 μl of areaction solution containing 10 ng of cDNA, 5 μl of 10×Ex taq buffer, 4μl of 2.5 mM each dNTP, 5 pmoles/sequence of each primer and 1 μl of Extaq (Takara)+Taq START antibody (Clontech) was run in 1 cycle at 94° C.for 3 min, 35 cycles of at 94° C. for 1 min, 50° C. for 1 min, and 72°C. for 1 min, and then 1 cycle at 72° C. for 6 min using a programmedtemperature control system PC-700 (ASTEK). As a result, a product ofabout 150-190 bp was amplified with each of primer pairs TMR1-TMRF1,TMR1-TMRF2, TMR2-TMRF1 and TMR2-TMRF2.

These PCR products were gel-purified and cloned into a vector pCRII(Invitrogen). These clones were sequenced to reveal that they containedtwo cDNAs, i.e. a cDNA encoding strictly the same amino acid sequence asdetermined in 1) (derived from pairs TMR2-TMRF1 and TMR2-TMRF2;especially, the cDNA derived from pair TMR2-TMRF2 is designated asTM100), and a cDNA encoding an amino acid sequence having a homology ofabout 75% to the amino acid sequence determined in 1) (derived frompairs TMR1-TMRF1, TMR1-TMRF2; especially, the cDNA derived from pairTMR1-TMRF1 is designated as TM75).

5) Screening of the Full-Length cDNA

The cDNA clones TM100 and TM75 obtained in 4) were excised from thevector and used as probes to screen the cDNA library of Pleurotuscornucopiae fruit body. In a square Petri dish (14×10 cm), about 20,000pfu of phage was plated with a host XL1-blue MRF′ according toinstructions given for a ZAP cDNA synthesis kit (Stratagene). The plaquewas brought into contact with Hybond-N+ nylon membrane filter (Amersham)to denature DNA by alkali as instructed for the membrane, andimmobilized on the membrane. The probes were ³²P-labeled using arediprime II™ DNA labelling system (Amersham). Hybridization wasperformed in 0.5 M NaHPO₄ (pH 7.2), 7% SDS, 1 mM EDTA at 65° C.overnight, followed by washing twice in 40 mM NaHPO₄ (pH 7.2), 1% SDS, 1mM EDTA at 65° C. for 20 minutes. Primary screening from about 160,000pfu of phage gave 600 positive clones with TM100 probe and 30 positiveclones with TM75 probe. Among them, 24 clones from TM100 probe and 12clones from TM75 probe were further subjected to secondary screening anda third screening also aimed at purifying the plaque, and all the clonesselected were in vivo excised as instructed for the ZAP cDNA synthesiskit (Stratagene). As a result, 18 clones from TM100 probe and 12 clonesfrom TM75 probe were recovered as cDNA integrated into the phagemidvector pBluescript SK. The insert length of these clones was identifiedby restriction endonuclease analysis.

6) Determining the Base Sequences

The total nucleotide sequence of the longest clone of each of the abovecDNA clones was determined. Initially, both 5′ and 3′ nucleotidesequences of the clone were determined using M13 primers (takara) on ABIPRISM Fluorescence Sequencer (Model 310 Genetic Analyzer, Perkin Elmer).

Then, the following primers were synthesized: TM100inRV: gTC AAg gCg TTACTC Tgg (SEQ ID NO: 14) based on the 5′ nucleotide sequence data of thelongest clone from TM100;

TM100inFW: CTg ggT gAg gAT CAC CTC (SEQ ID NO: 15) based on the 3′nucleotide sequence data of the same clone; TM75inRV: gAT gTC TAC gTgCCC TAC (SEQ ID NO: 16) based on the 5′ nucleotide sequence data of thelongest clone from TM75; andTM75inFW: ACg ACT CAg AgA AgA ACT g (SEQ ID NO: 17) based on the 3′nucleotide sequence data of the same clone; and used for sequencing.Thus, both DNA sequences of the longest clones from TM100 and TM75 weredetermined, so that the total nucleotide sequence was determined.

The results showed that the cDNA encoding Pleurotus cornucopiaeantifungal protein (from TM100 probe) consist of a total of 671 bases(SEQ ID NO: 1) encoding 143 amino acids (SEQ ID NO: 2). The N-terminalamino acid sequence determined from the purified protein corresponded tothe amino acid residues 8-76 of the amino acid sequence of SEQ ID NO: 2.The amino acid sequence of SEQ ID NO: 2 and 67 amino acids directlydetermined from the purified protein were totally identical except fortwo unknown amino acids (corresponding to W 65 and S 73 in the aminoacid sequence of SEQ ID NO: 2). This led to the conclusion that thecloned cDNA is derived from a gene encoding Pleurotus cornucopiaeantifungal protein.

The N-terminal sequence of the protein determined from the cDNA sequencewas not identical with the N-terminal sequence of the actually purifiedprotein, i.e. L (leucine) after 7 amino acids following the initiationcodon methionine was located at the N-terminus of the purified protein.This indicated that Pleurotus cornucopiae antifungal protein wastranslated as a precursor at first and then, the N-terminal leadersequence (7 amino acids) was truncated. The average molecular weight ofthe amino acid sequence of the putative mature protein (a sequence of136 amino acids 8-143 of SEQ ID NO: 2) was 15158.4 as determined using agene sequence analysis software program GENETYX-WIN ver 3.2 (SoftwareDevelopment Co., Ltd.), and the isoelectric point was calculated at6.22. This molecular weight agreed well with the estimation (15 kDa) ofthe purified protein by SDS-PAGE. In addition, two putativeglycosylation sites existed (N 21 and 71 of SEQ ID NO: 2).

On the other hand, the cDNA encoding a homolog of the Pleurotuscornucopiae antifungal protein (from TM75) consists of a total of 840bases (SEQ ID NO: 3) encoding 141 amino acids (SEQ ID NO: 4). The cDNAcontains three ATG codons at the 5′ end, but when translation startsfrom the first and second ATG codons, a termination codon appears closebehind them and only 12 and 31 amino acids can be encoded. Only whentranslation starts from the third ATG codon, 141 amino acids can beencoded. A termination codon TGA was located at 102 bp upstream of thisATG in the same reading frame. Thus, it is nearly certain that the thirdATG is an initiation codon.

The molecular weight of the putative mature amino acid sequence (asequence of 134 amino acids consisting of residues 8-141 of the aminoacid sequence of SEQ ID NO: 4) was estimated at 14732.2, and theisoelectric point was calculated at 8.62. One putative glycosylationsite existed (N 115 of SEQ ID NO: 4). The homology between the Pleurotuscornucopiae antifungal protein of the present invention and the homologwas 65.5% in amino acid and 64.5% in DNA (ORF 72.2%) as analyzed usingGENETYX-WIN.

Homology searches were performed through GenBank databases using BLASTfor the Pleurotus cornucopiae-derived antifungal protein of the presentinvention and the gene thereof as well as their homologs and proteinshaving an amino acid sequence encoded thereby. Database searches of theamino acid sequence of the Pleurotus cornucopiae-derived antifungalprotein of the present invention (total amino acid sequence of SEQ IDNO: 2) found homologous sequences such as streptavidin v2 ofStreptomyces violaceus (Accession No: Q53533, Bayer et al. (1995)Biochim Biophys Acta 1263: pp. 60-66.) and v1 (Accession No: Q53532),streptavidin of Streptomyces avidinii (Accession No.: P22629, Argaranaet al. (1986) Nucleic Acids Res 14: pp. 1871-1882), etc. The homologiesof these three sequences extend over 128 amino acids, and were 50%, 49%and 49%, respectively. Egg white avidin (Gope et al. (1987) NucleicAcids Res 15: pp. 3595-3606) and several avidin-related proteins(Keinanen et al. (1994) Eur J Biochem 220: pp. 615-621) were alsomatched at lower homology degrees. A core streptavidin mutant w79fChainB (Freitag et al. (1997) Protein Sci. 6: pp. 1157-1166) was alsomatched, which differs from streptavidin by only one amino acid and inwhich 36 N-terminal amino acids and 20 C-terminal amino acids ofstreptavidin are truncated. The homology was 51.7%.

These facts indicate that the present protein is a novel protein.Database searches of the amino acid sequence of the second Pleurotuscornucopiae-derived antifungal protein of the present invention (totalamino acid sequence of SEQ ID NO: 4) showed homology degrees of 50%, 48%and 48% to streptavidin v2, v1 and streptavidin, respectively.

However, similar database searches using the DNA sequence of a geneencoding the first Pleurotus cornucopiae-derived antifungal protein(71-502 of SEQ ID NO: 1) and the DNA sequence of a gene encoding thesecond Pleurotus cornucopiae-derived antifungal protein (226-651 of SEQID NO: 3) found only several sequences showing homology in a very shortrange (23 bp) but not the DNA sequence of streptavidin. This means thatthe DNA sequences encoding the novel proteins of the present inventionare not highly homologous to the DNA sequence of streptavidin on the DNAlevel.

The present antifungal protein was named “tamavidin” because it was anovel streptavidin-like protein purified from an edible mushroomPleurotus cornucopiae (Tamogitake). The gene derived from the purifiedprotein is called tam1, the protein having an amino acid sequenceencoded thereby is called tamavidin 1, a homolog of tam1 is called tam2, and the protein having an amino acid sequence encoded thereby iscalled tamavidin 2. When a genetic sequence analysis software programGENETYX-WIN ver 3.2 was used to analyze homology of the total amino acidsequences of the Pleurotus cornucopiae antifungal proteins of thepresent invention to streptavidin (which differs from streptavidins v2and v1 by only 9 amino acids and 1 amino acid, respectively), the aminoacid sequence of tamavidin 1 encoded by tam1 showed a homology (aminoacid identity) of 46.7% and the amino acid sequence of tamavidin 2encoded by tam2 showed 48.1%. The homology of the total DNA sequence(SEQ ID NOs: 1 and 3) to streptavidin was 53.8% (ORF 56.8%) for tam1 and51.0% (ORF 57.3%) for tam2. The homology of the Pleurotus cornucopiaeantifungal protein encoded by tam1 to egg white avidin was 31.2% inamino acid sequence and 42.4% in DNA sequence, and the homology of thePleurotus cornucopiae antifungal protein encoded by tam2 to egg whiteavidin was 36.2% in amino acid sequence and 41.8% in DNA sequence.

A molecular taxonomic tree of the amino acid sequences of the matureprotein regions of tamavidin 1, tamavidin 2, streptavidin, streptavidinV1, streptavidin V2 and avidin was prepared by the UPGMA method usingGENETYX-WIN. The results showed that tamavidin 1 and tamavidin 2 form athird group distinct from the streptavidin group and avidin group asshown in FIG. 7.

As compared with streptavidin, tamavidin 1 and tamavidin 2 of thepresent invention are truncated by 33 N-terminal amino acids, but allthe tryptophan (W) residues (Gitlin et al. (1988) Biochem. J. 256: pp.279-282) and tyrosine (Y) residues (Gitlin et al. (1990) Biochem. J.269: pp. 527-530) possibly involved in binding to biotin are conserved(Y 34 and 45 and W 82, 98 and 110 in the amino acid sequence of SEQ IDNO: 2, and Y 34 and 45 and W 80, 96 and 108 in the amino acid sequenceof SEQ ID NO: 4).

The average molecular weights of the regions supposed to be matureprotein regions (stretches 8-143 of the amino acid sequence of SEQ IDNO: 2, and 8-141 of the amino acid sequence of SEQ ID NO: 4) werecalculated at 15158.4 and 14732.2, respectively, close to the averagemolecular weights of mature streptavidin and mature avidin (16490.6 and14342.9, respectively). These facts strongly suggest that not onlytamavidin 1 encoded by tam1 but also tamavidin 2 encoded by tam2 is aprotein having biotin-binding affinity.

Example 4 Experiments of Abolishment of an Antifungal Activity byBiotinylation

The antifungal proteins of the present invention are novelstreptavidin-like proteins, suggesting that it binds to one of vitamins,D-biotin (vitamin H, Katayama Chemical). Rice blast is known to requirebiotin for its growth. These facts suggest that the present antifungalprotein binds to free biotin present in assay media to induce biotindeficiency in the media, with the result that the growth of rice blast(M. grisea) is inhibited.

FIG. 8 shows the results of experiments of abolishment of an antifungalactivity by addition of biotin. Specifically, spores of M. griseasuspended in ½ PD were placed in microtiter plates. Wells containing 50ng/ml or 1000 ng/ml of purified tamavidin 1, or wells containing 1000ng/ml of tamavidin1 and 100 ng/ml of biotin, or control wells containingno protein were prepared and incubated at 28° C. for 48 hours.

As a result, the extension of hyphae of M. grisea was fairly inhibitedin wells containing tamavidin 1 even at a concentration of 50 ng/ml asshown in FIG. 8. However, hyphae normally extended in wells containingboth tamavidin 1 (1000 ng/ml) and biotin and control wells. Thus, anantifungal activity of the present antifungal protein was actuallyabolished by excessively adding biotin into the assay media. This isprobably because a certain part of biotin excessively added bound tomost of tamavidin 1 used for the assay to inactivate its antifungalactivity.

Similar tests were performed on commercially available egg white avidin(Sigma) and streptavidin of streptomyces avidinii (Sigma) at aconcentration of 1000 ng/ml. The results showed that both proteins hadthe antifungal activity against M. grisea and the activity was abolishedby biotin (FIG. 8).

Example 5 Biotin-Binding Activity of Recombinant Tamavidin 2 Protein 1)Construction of an Expression Vector

Evaluations were performed as to find out whether or not tamavidin 2encoded by tam2 gene, which is a gene isolated as a homolog of tam1 geneof the present invention, practically shows biotin-binding activity.Specifically, tam2 gene was inserted into E. coli to express recombinanttamavidin 2 and was examined as to find out whether or not this proteinis purified on an iminobiotin column.

Initially, a primer pair was synthesized for amplifying the total ORF oftam2 gene obtained in Example 3 (bases 226-651 of SEQ ID NO: 3 in theSequence Listing) by PCR.

(SEQ ID NO: 18) TM75Bsp5: 5′-ACCAACATgTCAgACgTTCAA-3′ (SEQ ID NO: 19)TM75Hin3: 5′-ATgAAAgCTTTTACTTCAACCTCgg-3′.TM75Bsp5 contains a recognition site of restriction endonuclease BspLU11I (underlined) and TM75Hin3 contains a recognition site of restrictionendonuclease HindIII (underlined), respectively. These primers were usedto perform PCR on a plasmid containing tam2 gene (pBluescript, Example3(6)) as a template. Using a programmed temperature control systemPC-700 (ASTEK), 50 μl of a reaction solution containing 500 ng oftemplate plasmid DNA, 5 μl of 10× Pyrobest buffer, 4 μl of 2.5 mM eachdNTP, 10 pmoles of each primer and 0.5 μl of Pyrobest DNA polymerase(Takara) was run in 1 cycle at 94° C. for 3 min, 15 cycles of at 94° C.for 1 min, 50° C. for 1 min and 72° C. for 1 min, and then 1 cycle at72° C. for 6 min.

The resulting PCR product was double digested with restrictionendonucleases BspLU 111 (Roche) and HindIII (Takara) and subjected togel-purification. The E. coli expression vector used was pTrc99A(Pharmacia LKB). This vector was double-digested with NcoI (Takara) andHindIII and gel-purified and ligated with the PCR product treated withrestriction endonucleases as above, and then inserted into E. coli TB1.The nucleotide sequence of the inserted tam2 gene was confirmed.

2) Expression of the Recombinant Protein and Purification on a BiotinColumn

A single colony of E. coli bearing the expression vector pTrc99Acontaining TB1 tam2 was inoculated into LB medium containing anantibiotic ampicillin and precultured to reach about OD₆₀₀=0.5. Then,IPTG was added at a final concentration of 1 mM to induce proteinexpression and cells were cultured by shaking at 37° C. for further 4.5hours. The culture volume was 50 mL and a control without IPTG(Isopropyl-β-D (−)-thiogalactopyranoside, Wako Pure Chemical Industries)was also tested. Cultured cells were collected by centrifugation andstored at −80° C. until protein purification.

Tamavidin 2 was purified on iminobiotin referring to the method ofHofmann et al. Proc. Natl. Acad. Sci. USA 77: 4666-4668 (1980)). Cellswere suspended in 1.5 mL of buffer A (50 mM CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid, SIGMA), pH 11, 50 mM NaCl)and disrupted by sonication. After centrifugation, the supernatant wascollected as total soluble protein. A column having a diameter of 0.5 cmand a height of 5 cm was charged with 0.5 mL of 2-Iminobiotin-Agarose(SIGMA) and equilibrated with buffer A. The total soluble protein wasloaded on this iminobiotin agarose column. After the column was washedwith 5 mL of 50 mM CAPS pH 11, 500 mM NaCl, tamavidin 2 was eluted with1.5 mL of 50 mM NH₄OAC, pH 4.0. The total soluble protein and thefraction having passed through the column, the wash fraction and elutionfraction were subjected to SDS-PAGE electrophoresis on 15% PAGEL (ATTO).

After migration, the protein was stained with Coomassie Brilliant BlueR-250 (Wako Pure Chemical Industries). The results are shown in FIG. 9.As shown in FIG. 9, the total soluble protein fraction induced by 1 mMIPTG (T) showed a band around 15 kDa, which was not found in theuninduced fraction (C). This molecular weight agreed well with themolecular weight of 15467 deduced from 141 amino acids encoded by tam2gene.

In addition, this 15 kDa protein appeared in the fraction eluted with 50mM NH₄OAC, pH 4.0 (E), but not in the fraction having passed through thebiotin column (F) and the wash fraction of the column (W). The 15 kDaprotein formed a major element in the elution fraction. This resultshows that tamavidin 2 encoded by tam2 binds to biotin. The result alsoshows that it can be conveniently purified by the method shown above.The yield of recombinant tamavidin 2 expressed in E. coli obtained froma culture volume of 50 mL was about 1 mg.

EFFECTS

Formulations containing as an active ingredient a protein elementcharacterized by comprising a polypeptide consisting of the amino acidsequence of SEQ ID NO: 2 or 4 or a polypeptide consisting of a partialsequence thereof and capable of binding to biotin or a derivativethereof according to the present invention can be expected for use asantifungal agents.

Disease-resistant plants can also be created by integrating a DNA havingthe sequence of 71-502 or 92-502 of SEQ ID NO: 1 or a DNA having thesequence of 226-651 or 247-651 of SEQ ID NO: 3 of the present inventioninto an expression cassette containing a suitable constitutive promoterfunctional in a plant cell, an organ/time-specific promoter or aninducible promoter sequence responding to stress or pests and aterminator sequence functional in the plant cell and then introducingthe cassette into the plant cell to give a regenerated individual. Inthis case, a DNA sequence encoding a signal sequence for transporting tosmall cellular organs or a signal sequence for extracellular secretioncan also be linked to the DNA sequence encoding the antifungal proteinof the present invention.

The proteins of the present invention can be produced and prepared inmass in cells of E. coli, yeasts, plants, insects or animals such asXenopus by integrating a DNA sequence encoding the protein of thepresent invention into an expression vector capable of expressingforeign proteins in the cells. In this case, a DNA sequence encoding asignal sequence for transporting to small cellular organs or a signalsequence for extracellular secretion can also be linked to the DNAsequence encoding the antifungal protein of the present invention.

The strong interaction between the proteins of the present invention andbiotin can be applied to various analytic techniques that are currentlywidely used with streptavidin and avidin.

1. An isolated nucleic acid encoding a protein, wherein said protein hasthe amino acid sequence as set forth in SEQ ID NO:2; or said protein hasan amino acid sequence with 95% or more homology to said sequence.
 2. Anisolated nucleic acid encoding a protein, wherein said protein comprisesthe amino acid sequence 8-143 as set forth in SEQ ID NO:2; or saidprotein has an amino acid sequence with 95% or more homology to saidsequence.
 3. An isolated nucleic acid encoding a protein, wherein saidprotein has an amino acid sequence as set forth in SEQ ID NO:2; or saidprotein has an amino acid sequence with 90% or more homology to saidsequence and with the tyrosine (Y) residues at 34 and 45, and thetryptophan (w) residues at 82, 98 and 110 in the amino acid sequence ofSEQ ID NO:2 remaining unchanged.
 4. The isolated nucleic acid accordingto claim 3, wherein said protein has an amino acid sequence with 95% ormore homology to the amino acid sequence as set forth in SEQ ID NO:2. 5.An isolated nucleic acid encoding a protein, wherein said protein hasthe amino acid sequence 8-143 as set forth in SEQ ID NO:2; or saidprotein has an amino acid sequence with 90% or more homology to saidsequence and with the tyrosine (Y) residues at 34 and 45, and thetryptophan (w) residues at 82, 98 and 110 in the amino acid sequence ofSEQ ID NO:2 remaining unchanged.
 6. The isolated nucleic acid accordingto claim 5, wherein said protein has an amino acid sequence with 95% ormore homology to the amino acid sequence 8-143 as set forth in SEQ IDNO:2.
 7. The isolated nucleic acid of any one of claims 1-6, whereinsaid protein exhibits an antifungal activity.
 8. An isolated nucleicacid comprising nucleotides 71-502 of SEQ ID NO:1.
 9. An isolatednucleic acid having a nucleic acid sequence with 90% or more homology tonucleotides 71-502 of SEQ ID NO:1, or a nucleic acid which can hybridizeto the complement of said sequence under stringent conditions (5×SSC,5×Denhardt's solution, 1% SDS, 45-68° C. (without formamide) or 30-42°C. (50% formamide)).
 10. An isolated nucleic acid having a nucleic acidsequence with 95% or more homology to nucleotides 71-502 of SEQ ID NO:1.11. The isolated nucleic acid of claim 9 wherein the protein encoded bythe nucleic acid exhibits an antifungal activity.
 12. The isolatednucleic acid of claim 10 wherein the protein encoded by the nucleic acidexhibits an antifungal activity.
 13. A recombinant vector comprising thenucleic acid according to any one of claims 2 and
 5. 14. The recombinantvector according to claim 13, which is an expression vector.
 15. Thetransformant obtained by introducing the recombinant vector of claim 13.16. The transformant of claim 15, which is a klendusic plant.